1. Field of the Invention
The present invention relates to a method of determining an initial concentration of a target nucleic acid in a sample using real-time amplification data.
2. Description of the Related Art
Among many analytical methods of detecting and quantifying nucleic acids, PCR is one of the most commonly used methods, the principles of which are disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202.
Conventional PCR generally shows qualitative, end point results of an amplified DNA using electrophoresis, having drawbacks with regards to accurate quantitative detection of DNA. In order to overcome such drawbacks, a real-time PCR which makes DNA quantification possible using an optical signal agent such as a fluorescent dye and an optical detection system by detecting the optical (e.g., fluorescent) signal intensity directly proportional to the amplified DNA concentration has been developed.
A conventional method of quantifying an initial concentration of a nucleic acid using a nucleic acid amplification data is disclosed in U.S. Pat. Nos. 6,303,305 and 6,503,720. Here, a method of obtaining an initial concentration of a nucleic acid by amplifying a nucleic acid and obtaining a function representing the amount of amplified nucleic acid in each cycle of the amplification, and finding the nth derivative of the function is disclosed. U.S. Pat. No. 6,303,305 discloses a quantification method which takes the maximum value of the derivative as Ct (threshold cycle), and U.S. Pat. No. 6,503,720 discloses a quantification method which takes zero value of the derivative as Ct.
In addition, another conventional method of quantifying initial concentration of a nucleic acid using nucleic acid amplification data is disclosed in U.S. Patent Laid-open Publication No. 20020031768. Here, a method of quantifying nucleic acid concentration using a specific value of a derivative is provided.
In addition, in U.S. Patent Publication No. 20060047443, a method of determining an initial concentration of a nucleic acid using real-time nucleic acid amplification data based on a mathematical model of a correlation between the amount of nucleic acid amplified and the amplification cycle is disclosed, which, without using a derivative, calculates the initial concentration using the amplification cycle number or the amplification time corresponding to half the value of the intensity of maximum fluorescent signal subtracted by the background optical (e.g., fluorescent) signal, the amplification cycle or the amplification time corresponding to a maximum amplification efficiency, and the optical signal (e.g., fluorescence) intensity of a prior-to-amplification optical (e.g., fluorescent) signal of the nucleic acid subtracted by the background signal. The content disclosed in U.S. Patent Publication No. 20060047443 is included in the present invention in its entirety by reference.
However, there is still needs for a method of efficiently determining an initial concentration of nucleic acid in a sample.